The catalytic hairpin assembly induces a conformational change of the RNA aptamer enabled in situ imaging of circRNAs in tumor cells

Abstract

Circular RNAs (circRNAs) are ubiquitously expressed across all cell types and tissues, playing a pivotal role in regulating diverse biological processes and being implicated in various human cancers. Their inherent stability renders them highly promising for practical applications in diagnosing and treating numerous human diseases, especially as biomarkers. In this study, we have successfully engineered a novel luminescent RNA sensor utilizing catalytic hairpin assembly (CHA) to mediate aptamer conformational changes for both extracellular and intracellular circRNA detection. The CHA process is specifically triggered by the unique back-splice junction (BSJ) present in circRNA, enabling precise discrimination between circular and homologous linear RNA. Through rational design and modification of the RNA aptamer sequence to enhance its affinity for the CHA product, the modified RNA aptamer undergoes specific conformational changes that facilitate binding with small molecule dyes, thereby generating a pronounced fluorescence signal. Our approach exhibits robust performance and stability in complex biological systems, making it suitable for visualizing circRNA in tumor cells and detecting extracellular circRNA. Additionally, this method demonstrates excellent biocompatibility and minimal cytotoxicity in live cell imaging, along with superior specificity and sensitivity for target molecules. This technique offers a valuable tool for elucidating intricate physiological processes involving circRNA in live cells.